Fluorescent proteins, particularly green fluorescent proteins, and their uses are well known in the art. U.S. Pat. No. 5,491,084 discloses various uses of a green fluorescent protein, together with host cells having gene constructs encoding a GFP, and methods for selecting cells expressing a protein-of interest. U.S. Pat. Nos. 5,625,048 and 5,777,079 disclose modified GFPs having emission and excitation spectra different to those of wild-type GFPs. U.S. Pat. No. 5,804,387 discloses GFP mutants having modified excitation and emission spectra.
Determining in vivo protease activity is extremely desirable since proteases can have a significant effect upon cellular events. For example, the early stage of apoptosis (programmed cell death) is signified by protease (caspase) activity and so an assay for appropriate protease activity can be an assay for apoptosis. Apoptosis is an induced cell suicidal process that allows the biological organism to destroy damaged or unwanted cells in an orderly way (Kerr, J. F. R. et al., 1972, Br. J. Cancer, 26: 239–257), and because of this it is a very important cellular process. It plays a vital role in maintaining the normal physiological function in a variety of ways. For example, the process of apoptosis is used in the thymus to eliminate self-reactive T cells to avoid auto-immunity (Thompson, C. B., 1995, Science, 267: 1456). Furthermore, when DNA is damaged in a cell and cannot be repaired, the cell will enter apoptosis to avoid the formation of abnormalities in the tissue. Thus, failure of programmed cell death can cause cancer. On the other hand, excessive apoptosis can also cause great damage to the body; it is linked to many neural degenerative diseases such as Huntington disease and Alzheimer's disease.
In the last few years, a large number of studies have been conducted aiming to understand the process of apoptosis on a molecular basis. The signalling pathways that direct the programmed cell death process turns out to be very complicated (Ashkenazi, A. and Dixit, V. M., 1998, Science, 281: 1305–1308; Thornberry, N. A. and Lazebnik, Y., 1998, Science, 281: 1312–1316; Evan, G. and Littlewood, T., 1998, Science, 281: 1322–1326; Adams, J. M. and Cory, S., 1998, Science, 281: 1317–1322). There are many external signals that can trigger the initiation of apoptosis, including UV-irradiation, activation of the “death domain” via the TNF (tumour necrosis factor) receptor or CD95, treatment with hormones (e.g. glucocorticoid) or chemotherapy drugs (e.g. camptothecin) (Ashkenazi, A. and Dixit, V. M., 1998, supra; Nagata, S., 1997, Cell, 88: 355–365; Martin, S. J. and Cotter, T. G., 1991, Int. Radiat. Biol., 59: 1001–1016). As for the internal signals, it is known that apoptosis is the outcome of a programmed cascade of intracellular events, which are centred on the activation of a class of cysteine proteases called “caspases” (Thornberry, N. A. and Lazebnik, Y., 1998, supra). At present, the detailed molecular mechanisms by which apoptosis is regulated by the various internal and external signals are still not well understood.
The process of apoptosis can be detected at different stages. For example, some apoptosis assays are based on events that occur rather late in apoptosis, such as morphological changes of the cell, nuclear breakdown, and chromosomal fragmentation. Some assays can detect relatively early events such as the turn-over of certain phospholipids in the membrane (Martin, S. J. et al., 1995, J. Exp. Med., 182: 1545–1555). Alternatively, one can assay the activation of caspase-3 based on the fact that substrates of caspase-3 have a specific sensing and cleavage sequence (Nicholson, D. W., 1996, Nature Biotechnol., 14: 297–301). By linking a peptide encoding this substrate sequence to a fluorescent dye, one can detect a shift of the fluorescent properties of the dye when the peptide is cleaved by the activated caspase (CLONTECHniques, 1997, 12(1): 4–6). The activity of caspase-8 can be similarly assayed (www.clontech.com, ApoAlert Caspase Assay Kits). Since these methods utilize an optical detection, they are simple and quick. These methods do, however, have certain limitations. For example, the probes cannot penetrate the cell membrane, thus it is difficult to load the probes inside the cell. The assays are therefore done using crude cell lysates, i.e. not with whole (living) cells and not in vivo. Furthermore, the fluorescent change resulting from caspase cleavage involves mainly a shift of the emission spectrum (from blue to yellow-green) rather than an easier to assay total destruction of the fluorescence. In addition, its sensitivity is limited.